1. Field of the Invention
The present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods and compositions for the production of progenitor cells, such as hematopoietic progenitor cells and endothelial progenitor cells from embryonic stem cells. The invention also relates to kits for the production of progenitor cells and methods of screening for substances that promote differentiation of pluripotent stem cells.
2. Description of Related Art
In vitro, human embryonic stem cells are capable of indefinite proliferation in culture and are thus capable, at least in principle, of supplying cells and tissues for the replacement of failing or defective human tissue. Due to the significant medical potential of hematopoietic stem and progenitor cells, substantial work has been done to try to improve methods for the differentiation of hematopoietic progenitor cells from embryonic stem cells. In the human adult, a small number of hematopoietic stem cells present primarily in bone marrow produce heterogeneous populations of actively dividing hematopoietic (CD34+) progenitor cells that differentiate into all the cells of the blood system. The CD34+ marker is an imprecise definition of hematopoietic cells since other cell types, notably endothelial cells (blood vessels), also express CD34. Thus, other markers, such as the CD43 marker, may also be used to help identify hematopoietic progenitor cells (e.g., Kadaja-Saarepuu et al., 2007; Vodyanik et al., 2006). In an adult human, hematopoietic progenitors proliferate and differentiate to generate hundreds of billions of mature blood cells daily. Hematopoietic progenitor cells are also present in cord blood.
In addition to hematopoietic cells, it is useful to differentiate endothelial progenitor cells, and ultimately endothelial cells, from embryonic stem cells. Endothelial cells comprise the lining of the blood vessels and are important for a variety of processes in the body. For example, endothelial cells play roles in angiogenesis, regulation of blood pressure, blood clotting, inflammation, and filtration. Endothelial cells are a heterogeneous group of cells and may have a variety of characteristics depending upon vessel size, specification to a specific organ, and morphology. Some characteristics of endothelial cells include expression of CD31, CD105 (endoglin), and Willebrand factor (also called Factor VIII), as well as the ability to take up acetylated low density lipoprotein (ac-LDL).
Previous methods to promote the differentiation of pluripotent stem cells (PSCs) have required the formation of embryoid bodies (e.g., Chadwick et al., 2003) or the use of mouse feeder cells such as mouse embryonic fibroblasts (e.g., Wang et al., 2007). Unfortunately, these approaches have several drawbacks that may limit their clinical potential.
The formation of “embryoid bodies” (EBs), or clusters of growing cells, to induce differentiation generally involves in vitro aggregation of human pluripotent stem cells into EBs and allows for the spontaneous and random differentiation of human pluripotent stem cells into multiple tissue types that represent endoderm, ectoderm, and mesoderm origins. These three-dimensional EBs contain some fraction of progenitor cells that may be used to produce hematopoietic cells and endothelial cells. Unfortunately, methods for the formation of EBs are often inefficient and laborious, and the multiple complex steps involved in the formation and dissociation of EBs can make use of automation more difficult. For example, the process for forming EBs is inefficient in that it usually requires an entire colony of hematopoietic progenitor cells. Further, utilizing EBs requires complex methods such as the dissociation of embryoid bodies, which presents substantial problems for automation or large-scale automation.
The culture of human pluripotent cells with feeder cell lines, such as mouse fibroblasts, presents the risk of unexpected transformations that have previously been associated with interspecies exposure during co-culture. Since one of the objectives of human pluripotent stem cell cultures is to create tissues which can ultimately be transplanted into a human body, it is highly desirable that the stem cells are not exposed to cells of another species or to a medium that has been used to culture cells of another species. Accordingly, defining a culture condition that will permit the differentiation of human pluripotent stem cells into the hematopoietic lineage or endothelial lineage without a co-culture step of any kind is of great interest in the continued development of techniques for the production of human hematopoietic progenitor cells or endothelial progenitor cells from human pluripotent stem cells.
Using serum in differentiation medium can also present certain drawbacks and limitations. Serum, e.g., as used in Chadwick et al. (2003), is an animal product that may be used to provide nutrients to growing cells. However, the composition of a particular serum is uncertain across different batches, meaning that one batch of serum may have different growth factors or different concentrations of growth factors as compared to a different batch of the same type of serum. These uncertainties may contribute to the variable yield of hematopoietic cells produced across experiments performed under the same conditions. Additionally, the use of serum may present substantial regulatory issues during clinical development, further complicating commercialization.
There currently exists a clear need for efficient methods of differentiating pluripotent stem cells into hematopoietic progenitor cells or endothelial progenitor cells without either exposing the cells to material from another animal species or forming embryoid bodies. Further, there exists a need for a defined differentiation medium and conditions that allow further differentiation steps, give reproducible results, and do not require inclusion of serum or feeder cells.